Biology subjects

Harris, T.

Publications and source records attributed to Harris, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipid-associated architecture and divergent ammonium transport distinguish a bacterial Rhesus protein

Nitrification is a major process in the global nitrogen cycle, initiated by the oxidation of ammonia to nitrite. The ammonia-oxidising bacterium Nitrosomonas europaea catalyses this first and rate-limiting step and depends on ammonium both as an energy substrate and as a nitrogen source, making ammonium acquisition central to its physiology. Cellular ammonium transport is mediated by the Amt/Mep/Rh superfamily, whose members share a conserved fold despite divergent physiological roles. Remarkably, N. europaea lacks a canonical prokaryotic Amt transporter and instead expresses NeRh50, a Rh-family protein proposed to have been acquired from a eukaryotic lineage. Here, we determine a cryo-EM structure of NeRh50 that reveals tightly associated lipids at the monomer-monomer interfaces, including density consistent with an unusual inverted lipid orientation. Native mass spectrometry and molecular dynamics simulations further support persistent lipid association at these interfaces. NeRh50 also differs functionally from canonical AmtB, selectively mediating electrogenic ammonium transport with ~70-fold lower apparent affinity. Transport is enhanced under alkaline conditions but not by an imposed proton gradient, suggesting membrane-potential dependence. Together, these findings reveal divergence from canonical Amt proteins in both membrane-associated architecture and transport properties, providing insight into the functional and evolutionary diversification of the Amt/Mep/Rh superfamily.

biochemistry↗

bioRxiv2026

Neuropixels 1536 Channel Quad Base probe reveals brain-wide communication underlying flexible sensorimotor sequences

The brain orchestrates billions of neurons spanning hundreds of regions to drive behavior. High-density electrophysiology has provided insights into this process, but monitoring brain-wide patterns of spiking activity demands new tools. Here we present Neuropixels 2.0 Quad Base, a 1536-channel probe with four times the simultaneous recording capacity of state-of-art Neuropixels 2.0. Using two Quad Base probes, we performed 3072-channel recordings in mice trained to produce complex sequences of goal-directed licks. We decoded behavioral kinematics from neural populations across more than 20 brain regions and identified the spatiotemporal emergence of motor decision signals. The probes high channel count further revealed expanded inter-neuronal functional subnetworks and latent dynamics across multiple areas--features significantly undersampled when channels were subsampled to mimic standard Neuropixels 2.0. Neuropixels 2.0 Quad Base offers a powerful tool to explore neural encoding of cognition and behavior at grand scale.

neuroscience↗

bioRxiv2026

Intelliwaste: NMR of 13C-labeled Spent Media Enables Non-Invasive Metabolic Fingerprinting of Pluripotent Stem Cells and LIS1-Associated Neuropathology

Metabolic dysregulation is increasingly recognized as a key contributor to neurodevelopmental disorders. Here, we present Intelliwaste, a non-invasive, cost-effective method for profiling carbon metabolism in pluripotent stem cells and brain organoids using 13C-labeled metabolites and 1H and 13C NMR spectroscopy. This approach enables longitudinal analysis of extracellular fluxes without disrupting cell viability. We apply Intelliwaste to human embryonic stem cells (hESCs) cultured in a defined media enriched with >95% 13C1-Glucose. Under these conditions, 13C3-lactate emerged as the most abundant labeled product, with 20-50-fold lower fluxes to 13C3-alanine, 13C2-acetate, 13C3-serine, and 13C3-pyruvate, and 100-300-fold lower fluxes to 13C1-formate and multiple 13C-labeled glutamate species. These profiles allow for precise quantification of fractional metabolic isotopic labeling and glucose-derived carbon flow. To demonstrate biological utility, we first examine the effect of L-glutamine omission, which selectively reduces 13C3-alanine/13C3-lactate and 13C4-glutamate/13C3-lactate flux ratios, while the 13C3-Glutamate/13C3-Lactate and 13C2-Glutamate/13C3-Lactate flux ratios remained unchanged. These findings suggest a specific role for extracellular glutamine in modulating the activity of alanine aminotransferase and pyruvate carboxylase. We then characterized LIS1 mutant hESCs--a model of lissencephaly--and observed significantly increased flux ratios involving 13C4-, 13C3-, and 13C2-glutamate relative to 13C3-lactate, indicating enhanced glutamate production via the TCA cycle. These findings establish Intelliwaste as a powerful tool for metabolic profiling in the study of human neurodevelopment and disease. Its non-destructive nature makes it particularly well-suited for tracking metabolic changes during differentiation and in patient-derived organoid models of neurological disorders.

cell biology↗

bioRxiv2026

SCREWx: A Screwless, Chronic, Recoverable, and Lightweight Neuropixels fixture for freely-moving rodents

High-density Neuropixels probes enable the study of large neural populations with single-cell and sub-millisecond resolution. While single-probe and acute head-fixed experiments have yielded critical scientific insights, understanding the neural mechanisms underlying many complex behaviors requires simultaneous multi-region recordings in freely moving, chronically implanted animals. Various probe fixtures have been developed to enable high-density recording, but existing designs impose critical limitations: their substantial weight restricts the maximum probe count that smaller animals can support, their bulky dimensions constrain the proximity of targeted brain regions, and their complex assembly risks damaging the probe during insertion and recovery. In this paper, we present a lightweight, fully 3D-printable, compact, and screwless fixture for chronic Neuropixels implants in freely moving rodents that features simple mechanisms for stable implantation and safe extraction. Our fixture design enables stable, high-yield single-unit recordings for months-long experiments, along with an 83% successful probe extraction rate. This fixture design provides a robust and accessible solution for long-term, multi-probe chronic Neuropixels recordings, increasing experimental throughput and enabling more complex experimental designs to investigate brain-wide neural dynamics.

neuroscience↗

bioRxiv2024

Altered Extracellular Matrix Structure and Elevated Stiffness in a Brain Organoid Model for Disease

The viscoelasticity of tissues impacts their shape, as well as the growth and differentiation of their cells. Nevertheless, little is known about changes in viscoelastic properties during brain malformations. Lissencephaly is a severe malformation of cortical development caused by LIS1 mutations, which results in a lack of cortical convolutions. Here, we show that human-derived brain organoids with LIS1 mutation are stiffer than control ones at multiple developmental times. This stiffening is accompanied by abnormal ECM expression and organization, as well as elevated water content, as measured by diffusion-weighted MRI. Proteolytic cleavage of ECM components by short-term treatment with the catalytic subunit of MMP9 reduced the stiffening and water diffusion levels of mutated organoids to control levels. Finally, based on the molecular and rheological properties obtained, we generated a computational microstructure mechanical model that can successfully predict mechanical changes that follow differential ECM localization and integrity in the developing brain. Overall, our study reveals that LIS1 is essential for the expression and organization of ECM proteins during brain development, and its mutation leads to a substantial viscoelastic change. To our knowledge, this is the first study to elucidate how tissue mechanics change in disease states using human brain organoids.

developmental biology↗

bioRxiv2023

Brain-wide neural activity underlying memory-guided movement

Behavior requires neural activity across the brain, but most experiments probe neurons in a single area at a time. Here we used multiple Neuropixels probes to record neural activity simultaneously in brain-wide circuits, in mice performing a memory-guided directional licking task. We targeted brain areas that form multi-regional loops with anterior lateral motor cortex (ALM), a key circuit node mediating the behavior. Neurons encoding sensory stimuli, choice, and actions were distributed across the brain. However, in addition to ALM, coding of choice was concentrated in subcortical areas receiving input from ALM, in an ALM-dependent manner. Choice signals were first detected in ALM and the midbrain, followed by the thalamus, and other brain areas. At the time of movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

neuroscience↗

bioRxiv2023

Large-scale brain-wide neural recording in nonhuman primates

High-density, integrated silicon electrodes have begun to transform systems neuroscience, by enabling large-scale neural population recordings with single cell resolution. Existing technologies, however, have provided limited functionality in nonhuman primate species such as macaques, which offer close models of human cognition and behavior. Here, we report the design, fabrication, and performance of Neuropixels 1.0-NHP, a high channel count linear electrode array designed to enable large-scale simultaneous recording in superficial and deep structures within the macaque or other large animal brain. These devices were fabricated in two versions: 4416 electrodes along a 45 mm shank, and 2496 along a 25 mm shank. For both versions, users can programmatically select 384 channels, enabling simultaneous multi-area recording with a single probe. We demonstrate recording from over 3000 single neurons within a session, and simultaneous recordings from over 1000 neurons using multiple probes. This technology represents a significant increase in recording access and scalability relative to existing technologies, and enables new classes of experiments involving fine-grained electrophysiological characterization of brain areas, functional connectivity between cells, and simultaneous brain-wide recording at scale.

neuroscience↗

bioRxiv2022

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

BackgroundFetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. MethodsIn a case control study of murine and human control and FGR placentae, we implied MR imaging, IHC and metabolomics to assess the levels of BPGM and 2,3 BPG to elucidate the impact of maternal gestational hypoxia, and the molecular mechanisms underlying human FGR. ResultsWe show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM, was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. ConclusionsThis study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. FundingThis work was supported by the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research (to MN) and by the Israel Science Foundation KillCorona grant 3777/19 (to MN, MK). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/507932v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@500160org.highwire.dtl.DTLVardef@8a68a9org.highwire.dtl.DTLVardef@dd60c3org.highwire.dtl.DTLVardef@1fbb209_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2021

A mechanistic switch from selective transporter to an ion channel impairs the filamentation signalling capability of ammonium transceptors in yeast

Ammonium translocation through biological membranes by the ubiquitous Amt-Mep-Rh family of transporters plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of these transporters, forming the familys characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology, in vivo yeast functional complementation and in silico molecular dynamics simulations, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs the specificity in Amt-Mep transporters but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We further show that both mechanisms coexist in fungal Mep2 Twin-His variants, disrupting the transceptor function and so inhibiting the filamentation process. These data strongly support a transport mechanism-mediated signalling process in the long-standing debate on the sensory function of Mep2-like transporters.

microbiology↗

bioRxiv2021

Allelic variation in Class I HLA determines pre-existing memory responses to SARS-CoV-2 that shape the CD8+ T cell repertoire upon viral exposure

Effective presentation of antigens by HLA class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8+ T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR /{beta} sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure. One-Sentence SummaryWe perform a unified, multi-omic characterization of the CD8+ T cell response to SARS-CoV-2, revealing pre-existing immunity conditioned by HLA genotype.

immunology↗

bioRxiv2021

A simple and robust method for automating analysis of naïve and regenerating peripheral nerves

BackgroundManual axon histomorphometry (AH) is time- and resource-intensive, which has inspired many attempts at automation. However, there has been little investigation on implementation of automated programs for widespread use. Ideally such a program should be able to perform AH across imaging modalities and nerve states. AxonDeepSeg (ADS) is an open source deep learning program that has previously been validated in electron microscopy. We evaluated the robustness of ADS for peripheral nerve axonal histomorphometry in light micrographs prepared using two different methods. MethodsAxon histomorphometry using ADS and manual analysis (gold-standard) was performed on light micrographs of naive or regenerating rat median nerve cross-sections prepared with either toluidine-resin or osmium-paraffin embedding protocols. The parameters of interest included axon count, axon diameter, myelin thickness, and g-ratio. ResultsManual and automatic ADS axon counts demonstrated good agreement in naive nerves and moderate agreement on regenerating nerves. There were small but consistent differences in measured axon diameter, myelin thickness and g-ratio; however, absolute differences were small. Both methods appropriately identified differences between naive and regenerating nerves. ADS was faster than manual axon analysis. ConclusionsWithout any algorithm retraining, ADS was able to appropriately identify critical differences between naive and regenerating nerves and work with different sample preparation methods of peripheral nerve light micrographs. While there were differences between absolute values between manual and ADS, ADS performed consistently and required much less time. ADS is an accessible and robust tool for AH that can provide consistent analysis across protocols and nerve states.

neuroscience↗

bioRxiv2021

Emergence of a smooth interface from growth of a dendritic network against a mechanosensitive contractile material

Structures and machines require smoothening of raw materials. Self-organized smoothening guides cell and tissue morphogenesis, and is relevant to advanced manufacturing. Across the syncytial Drosophila embryo surface, smooth interfaces form between expanding Arp2/3-based actin caps and surrounding actomyosin networks, demarcating the circumferences of nascent dome-like compartments used for pseudo-cleavage. We found that smoothening of the actomyosin interfaces requires Arp2/3 in vivo. To dissect the physical basis of this requirement, we reconstituted the interacting networks using node-based models. When actomyosin networks were simulated with clearances instead of Arp2/3 networks, rough boundaries persisted with low levels of myosin contractility. With addition of expanding Arp2/3 networks, network-network interfaces failed to smoothen, but accumulated myosin nodes and tension. After incorporating actomyosin mechanosensitivity, Arp2/3 network growth induced local contractility and smoothening of the interfaces, effects also evident in vivo. In this way, a smooth structure can emerge from the lateral interaction of irregular starting materials.

cell biology↗

bioRxiv2020

IL-12 signaling promotes TET2-mediated DNA demethylation during CD8 T cell effector differentiation

CD8 T cell memory differentiation endows T cells with an ability to rapidly induce effector functions upon pathogen re-encounter. While it is well established that substantial epigenetic remodeling occurs during the effector stage of the immune response, the signaling events that imprint CD8 T cells with these stable epigenetic programs are not well-defined. To gain insight into the signaling determinants of effector-associated epigenetic programming among CD8 T cells, we explored the role of IL-12 in the imprinting of IFNg expression during human CD8 T cell priming. We observed that TCR-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promotor. However, TCR stimulation in the presence of the inflammatory cytokine, IL-12, resulted in significant and stable demethylation of the IFNg locus that was commensurate with an increase in IFNg expression. We further show that IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent passive demethylation in an ex vivo human CAR T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector epigenetic programming in CD8 T cells during the primary immune response and serve as proof of concept that signal 3 cytokines can be used to guide the induction of epigenetically regulated traits among T cells used for adoptive immunotherapies.

immunology↗

bioRxiv2020

Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an "open" conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

biochemistry↗

bioRxiv2020

Predicting ovulation from brain connectivity: Dynamic causal modelling of the menstrual cycle.

Longitudinal menstrual cycle research allows the assessment of sex hormones effects on brain organization in a natural framework. Here, we used spectral dynamic causal modelling (spDCM) in a triple network model consisting of the default mode, salience and executive central networks (DMN, SN, and ECN), in order to address the changes in effective connectivity across the menstrual cycle. Sixty healthy young women were scanned three times (menses, pre-ovulatory and luteal phase) and spDCM was estimated for a total of 174 scans. Group level analysis using Parametric empirical Bayes showed lateralized and anterior-posterior changes in connectivity patterns depending on the cycle phase and related to the endogenous hormonal milieu. Right before ovulation the left insula recruited the frontoparietal network, while the right middle frontal gyrus decreased its connectivity to the precuneus. In exchange, the precuneus engaged bilateral angular gyrus, decoupling the DMN into anterior/posterior parts. During the luteal phase, bilateral insula engaged to each other decreasing the connectivity to parietal ECN, which in turn engaged the posterior DMN. Remarkably, the specific cycle phase in which a woman was in could be predicted by the connections that showed the strongest changes. These findings further corroborate the plasticity of the female brain in response to acute hormone fluctuations and have important implications for understanding the neuroendocrine interactions underlying cognitive changes along the menstrual cycle.

neuroscience↗

bioRxiv2020

Coadaptation of the chemosensory system with voluntary exercise behavior in mice

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process, strongly suggesting the chemosensory receptors as quantitative trait loci (QTL) for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals.

animal behavior and cognition↗

bioRxiv2020

Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection

Microglia, the resident immune cells of the brain parenchyma, are thought to be first-line defenders against CNS infections. We sought to identify specific roles of microglia in the control of the eukaryotic parasite Toxoplasma gondii, an opportunistic infection that can cause severe neurological disease. In order to identify the specific function of microglia in the brain during infection, we sorted microglia and infiltrating myeloid cells from infected microglia reporter mice. Using RNA-sequencing, we find strong NF-{kappa}B and inflammatory cytokine signatures overrepresented in blood-derived macrophages versus microglia. Interestingly, we also find that IL-1 is enriched in microglia and IL-1{beta} in macrophages, which was also evident at the protein level. We find that mice lacking IL-1R1 or IL-1, but not IL-1{beta}, have impaired parasite control and immune cell infiltration specifically within the brain. Further, by sorting purified populations from infected brains, we show that microglia, not peripheral myeloid cells, release IL-1 ex vivo. Finally, using knockout mice as well as chemical inhibition, we show that ex vivo IL-1 release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in immune infiltration into the brain and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia specifically can release the alarmin IL-1, a cytokine that promotes neuroinflammation and parasite control.

immunology↗

bioRxiv2018

IL-10 and ICOS differentially regulate T cell responses in the brain during chronic Toxoplasma gondii infection

Control of chronic CNS infection with the parasite Toxoplasma gondii requires an ongoing T cell response in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokine exclusively during the chronic phase of infection we blocked IL-10 receptor (IL-10R). Blockade was associated with widespread changes in the inflammatory response, including increased antigen presenting cell (APC) activation, expansion of CD4+ T cells, and increased neutrophil recruitment to the brain, consistent with previous reports. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS (inducible T cell costimulator), a molecule that promotes IL-10 production in many systems. Unexpectedly, ICOS-ligand (ICOSL) blockade led to a local expansion of effector T cells in the inflamed brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. Specifically, we observed increased expression of IL-2 associated signaling molecules, including CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cell populations or neutrophil recruitment to the brain Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection.

immunology↗